Suppression of LPS-induced inflammatory and NF-κB responses by anomalin in RAW 264.7 macrophages

The treatment of inflammatory diseases today is largely based on interrupting the synthesis or action of the mediators that drive the host's response to injury. It is on the basis of this concept that most of the anti-inflammatory drugs have been developed. In our continuous search for novel anti-inflammatory agents from traditional medicinal plants, Saposhnikovia divaricata has been a focus of our investigations. Anomalin, a pyranocoumarin constituent of S. divaricata, exhibits potent anti-inflammatory activity. To clarify the cellular signaling mechanisms underlying the anti-inflammatory action of anomalin, we investigated the effect of anomalin on the production of inflammatory molecules in LPS-stimulated murine macrophages. The anomalin dose-dependently inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA and protein expression in LPS-stimulated RAW 264.7 macrophage. Molecular analysis using quantitative real time polymerase chain reaction (qRT-PCR) revealed that several pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), were reduced by anomalin, and this reduction correlated with the down-regulation of the NF-κB signaling pathway. In addition, anomalin suppressed the LPS-induced phosphorylation and degradation of IκBα. To further study the mechanisms underlying its anti-inflammatory activity, an electrophoretic mobility shift assay (EMSA) using a (32) P-labeled NF-κB probe was conducted. LPS-induced NF-κB DNA binding was drastically abolished by anomalin. The present data suggest that anomalin is a major anti-inflammatory agent and may be a potential therapeutic candidate for the treatment of inflammatory disorders.     

Casuarinin suppresses TNF-α-induced ICAM-1 expression via blockade of NF-κB activation in HaCaT cells

Hippophae rhamnoides has been extensively used in oriental traditional medicines for treatment of asthma, skin diseases, gastric ulcers, and lung disorders. In this study, we isolated casuarinin from the leaves of H.rhamnoides and examined the effect of casuarinin on the TNF-α-induced ICAM-1 expression in a human keratinocytes cell line HaCaT. Pretreatment with casuarinin inhibited TNF-α-induced protein and mRNA expression of ICAM-1 and subsequent monocyte adhesiveness in HaCaT cells. Casuarinin significantly inhibited TNF-α-induced NF-κB activation. In addition, casuarinin inhibited activation of ERK and p38 MAPK in a dose-dependent manner. Furthermore, pretreatment with casuarinin decreased TNF-α-induced pro-inflammatory mediators, such as IL-1β, IL-6, IL-8, and MCP-1. These results demonstrated that casuarinin exerts its anti-inflammatory activity by suppressing TNF-α-induced expression of ICAM-1 and pro-inflammatory cytokines/chemokines via blockage of activation of NF-κB and ERK/p38 MAPK and can be used as a therapeutic agent against inflammatory skin diseases.     

Anti-inflammatory activity of myricetin from Diospyros lotus through suppression of NF-κB and STAT1 activation and Nrf2-mediated HO-1 induction in lipopolysaccharide-stimulated RAW264.7 macrophages

Diospyros lotus is traditionally used for the treatment of diabetes, diarrhea, tumor, and hypertension. The purpose of this study was to investigate the anti-inflammatory effect and underlying molecular mechanisms of myricetin in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Myricetin dose-dependently suppressed the production of pro-inflammatory mediators (NO, iNOS, PGE₂, and COX-2) in LPS-stimulated RAW264.7 macrophages. Myricetin administration decreased the production of NO, iNOS, TNF-α, IL-6, and IL-12 in mice. Myricetin decreased NF-κB activation by suppressing the degradation of IκBα, nuclear translocation of p65 subunit of NF-κB, and NF-κB DNA binding activity in LPS-stimulated RAW264.7 macrophages. Moreover, myricetin attenuated the phosphorylation of STAT1 and the production of IFN-β in LPS-stimulated RAW264.7 macrophages. Furthermore, myricetin induced the expression of HO-1 through Nrf2 translocation. In conclusion, these results suggest that myricetin inhibits the production of pro-inflammatory mediators through the suppression of NF-κB and STAT1 activation and induction of Nrf2-mediated HO-1 expression in LPS-stimulated RAW264.7 macrophages.     

Casuarinin suppresses TNF-α-induced ICAM-1 expression via blockade of NF-κB activation in HaCaT cells

The GRB2 associated binder 1 (GAB1) is an essential docking/adaptor protein for transmitting intracellular signals of the MET tyrosine kinase receptor activated by hepatocyte growth factor/scatter factor (HGF/SF). We found that in response to hours of HGF/SF treatment, the GAB1 protein level is degraded by a mechanism involving MET activity and the proteasomal machinery. We also showed that GAB1 is both multi- and poly-ubiquitinated in a CBL-dependent manner. A long term exposure to HGF/SF caused a more sustained down-regulation of GAB1 than of MET, associated with a loss of reactivation of the ERK MAP kinases to subsequent acute ligand treatment. These data demonstrate that GAB1 is ubiquitinated by CBL and degraded by the proteasome, and plays a role in negative-feedback regulation of HGF/SF–MET signaling.     

BRCA-1 depletion impairs pro-inflammatory polarization and activation of RAW 264.7 macrophages in a NF-κB-dependent mechanism

Molecular and Cellular Biochemistry - Inadequate migration and invasion of the trophoblast cells during embryo implantation is one of the reasons for pregnancy-related complications such as...     

The glucosamine-mediated induction of CHOP reduces the expression of inflammatory cytokines by modulating JNK and NF-κB in LPS-stimulated RAW264.7 cells

Macrophages secrete inflammatory cytokines and mono-nitrogen oxide (NO), and play crucial roles in inflammation in early-stage rheumatoid arthritis (RA). This study investigated whether glucosamine hydrochloride (GlcN), a nonsteroidal anti-inflammatory agent (NSAID) widely used to treat arthritis, affects the expression of inflammatory cytokines via the unfolded protein response (UPR) in lipopolysaccharide (LPS)-stimulated mouse macrophages (RAW264.7 cells). Pretreatment with GlcN reduced the expression of inflammatory cytokines and inhibited cell differentiation. Moreover, GlcN treatment increased the expression of CHOP and BiP/Grp78, the UPR target genes, in the presence or absence of LPS. Indeed, knockdown of CHOP using siRNAs prevented the GlcN-mediated reduction of inflammatory cytokines in LPS-stimulated RAW264.7 cells. Finally, we found that GlcN-mediated induction of CHOP reduced the phosphorylation of JNK and NF-?B in LPS-stimulated RAW264.7 cells. Combined, these results suggest that the GlcN-mediated induction of CHOP negatively regulates the inflammatory response by modulating JNK and NF-?B in LPS-stimulated RAW264.7 cells.     

Casuarinin suppresses TARC/CCL17 and MDC/CCL22 production via blockade of NF-κB and STAT1 activation in HaCaT cells

Casuarinin is a naturally occurring tannin that is isolated from the leaves of Hippophae rhamnoides. It has been shown to have anti-oxidant, anti-cancer, anti-viral, and anti-inflammatory activities. The aim of this study was to investigate the possible mechanism by which casuarinin inhibits TNF-α/IFN-γ-induced Th2 chemokines expression in the human keratinocytes cell line HaCaT. We found that casuarinin suppressed TNF-α/IFN-γ-induced expression of TARC and MDC mRNA and protein in HaCaT cells. Casuarinin significantly inhibited TNF-α/IFN-γ-induced activation of NF-κB, STAT1, and p38 MAPK. Furthermore, we observed that p38 MAPK contributes to inhibition of TNF-α/IFN-γ-induced TARC and MDC production by blocking NF-κB and STAT1 activation in HaCaT cells. Taken together, these results suggest that casuarinin may exert anti-inflammatory responses by suppressing TNF-α/IFN-γ-induced expression of TARC and MDC via blockage of p38 MAPK activation and subsequent activation of NF-κB and STAT1. We propose that it could therefore be used as a therapeutic agent against inflammatory skin diseases.     

Dalesconols B inhibits lipopolysaccharide induced inflammation and suppresses NF-κB and p38/JNK activation in microglial cells

Therapeutic strategies designed to inhibit the activation of microglia may lead to significant advancement in the treatment of most neurodegenerative diseases. Dalesconols B, also termed as TL2, is a newly found polyketide from a mantis-associated fungus and has been reported to exert potent immunosuppressive effects. In the present study, the anti-inflammatory effects of TL2 was investigated in lipopolysaccharide (LPS)-treated BV2 microglia and primary microglia cells. Our observations indicated that pretreatment with TL2 significantly inhibited the production of NO and PGE2 and suppressed the expression of pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), COX-2, TNF-α, IL-1β, IL-6, MCP-1 and MIP-1α in LPS-stimulated BV2 microglia. The nuclear translocation of NF-κB and the phosphorylation level of Akt, p38 and JNK MAP kinase pathways were also inhibited by TL2 in LPS-treated BV2 microglia. Moreover, TL2 also decreased Aβ-induced production of TNF-α, IL-1β and IL-6 in BV2 microglia. Additionally, TL2 protected primary cortical neurons against microglia-mediated neurotoxicity. Overall, our findings suggested that TL2 might be a promising therapeutic agent for alleviating the progress of neurodegenerative diseases associated with microglia activation.     

β-Glucan from Saccharomyces cerevisiae reduces lipopolysaccharide-induced inflammatory responses in RAW264.7 macrophages

Background

β-Glucans obtained from fungi, such as baker's yeast (Saccharomyces cerevisiae)-derived β-glucan (BBG), potently activate macrophages through nuclear factor κB (NFκB) translocation and activation of its signaling pathways. The mechanisms by which β-glucans activate these signaling pathways differ from that of lipopolysaccharide (LPS). However, the effects of β-glucans on LPS-induced inflammatory responses are poorly understood. Here, we examined the effects of BBG on LPS-induced inflammatory responses in RAW264.7 mouse macrophages.

Methods

We explored the actions of BBG in RAW264.7 macrophages.

Results

BBG inhibited LPS-stimulated nitric oxide (NO) production in RAW264.7 macrophages by 35–70% at concentrations of 120–200 μg/ml. BBG also suppressed mRNA and protein expression of LPS-induced inducible NO synthase (iNOS) and mitogen-activated protein kinase phosphorylation, but not NFκB activation. By contrast, a neutralizing antibody against dectin-1, a β-glucan receptor, did not affect BBG-mediated inhibition of NO production. Meanwhile, BBG suppressed Pam3CSK-induced NO production. Moreover, BBG suppressed LPS-induced production of pro-and anti-inflammatory cytokines, including interleukin (IL)-1α, IL-1ra, and IL-27.

Conclusions

Our results indicate that BBG is a powerful inhibitor of LPS-induced NO production by downregulating iNOS expression. The mechanism involves inactivation of mitogen-activated protein kinase and TLR2 pathway, but is independent of dectin-1.

General significance

BBG might be useful as a novel agent for the chemoprevention of inflammatory diseases.     

Magnesium isoglycyrrhizinate suppresses LPS-induced inflammation and oxidative stress through inhibiting NF-κB and MAPK pathways in RAW264.7 cells

Magnesium Isoglycyrrhizinate (MgIG), a novel molecular compound extracted from licorice root, has exhibited greater anti-inflammatory activity and hepatic protection than glycyrrhizin and β-glycyrrhizic acid. In this study, we investigated the anti-inflammatory effect and the potential mechanism of MgIG on Lipopolysaccharide (LPS)-treated RAW264.7 cells. MgIG down-regulated LPS-induced pro-inflammatory mediators and enzymes in LPS-treated RAW264.7 cells, including TNF-α, IL-6, IL-1β, IL-8, NO and iNOS. The generation of reactive oxygen species (ROS) in LPS-treated RAW264.7 cells was also reduced. MgIG attenuated NF-κB translocation by inhibiting IKK phosphorylation and IκB-α degradation. Simultaneously, MgIG also inhibited LPS-induced activation of MAPKs, including p38, JNK and ERK1/2. Taken together, these results suggest that MgIG suppresses inflammation by blocking NF-κB and MAPK signaling pathways, and down-regulates ROS generation and inflammatory mediators.     

Autophagy in the intestinal epithelium reduces endotoxin-induced inflammatory responses by inhibiting NF-κB activation

Autophagy is a lysosomal degradation pathway that is essential for survival, differentiation, development and homeostasis. There is growing evidence that impaired autophagy leads to the pathogenesis of diverse diseases. However, the role of autophagy in intestinal epithelium is not clearly understood, although previous studies have pointed out the possibility for the relationships of autophagy with bowel inflammation. In this study, we investigated the involvement of autophagy in intestinal epithelium with inflammatory responses. We generated the mice with a conditional deletion of Atg7, which is one of the autophagy regulated gene, in intestinal epithelium. In Atg7-deficient small intestinal epithelium, LPS-induced production of TNF-α and IL-1β mRNA was enhanced in comparison to the control small intestinal tissues. In addition, the degree of LPS-induced activation of NF-κB was promoted in Atg7-deficient intestinal epithelium. These results demonstrate that autophagy can attenuate endotoxin-induced inflammatory responses in intestinal epithelium resulting in the maintenance of intestinal homeostasis.     

Palmitate induces SHIP2 expression via the ceramide-mediated activation of NF-κB,and JNK in skeletal muscle cells

Aims

Elevated plasma free fatty acids impair the insulin signaling by induction of the expression of protein phosphatases. However, the effect of palmitate on SH2-containing inositol 5′-phosphatase 2 (SHIP2) expression has not been investigated. Here we investigated the effects of palmitate on SHIP2 expression and elucidated the underlying mechanisms in skeletal muscle cells.

Main methods

SHIP2 mRNA and protein levels were measured in C2C12 myotubes exposed to palmitate. Specific inhibitors were used to identify the signaling pathways involved in SHIP2 expression.

Key findings

The results showed that 0.5 mM palmitate significantly upregulates the mRNA and protein levels of SHIP2 in C2C12 cells. To address the role of palmitate intracellular metabolites in SHIP2 expression, the myotubes were treated with palmitate in the presence of ceramide and diacylglycerol synthesis inhibitors. The results demonstrated that only ceramide synthesis inhibition could prevent palmitate-induced SHIP2 expression in these cells. In addition, the incubation of muscle cells with different concentrations of C2-ceramide dose-dependently enhanced SHIP2 expression. Furthermore, the inhibition of both JNK and NF-κB pathways could prevent ceramide-induced SHIP2 expression in myotubes.

Significance

These findings suggest that palmitate contributes to SHIP2 overexpression in skeletal muscle via the mechanisms involving the activation of ceramide-JNK and NF-κB pathways.     

Quercetin suppresses proinflammatory cytokines production through MAP kinases and NF-κB pathway in lipopolysaccharide-stimulated macrophage

Quercetin is a flavonoid molecule ubiquitous in nature and functions as an anti-oxidant and anti-inflammatory agent with little toxicity in vivo and in vitro. Dose- and time-dependent effect of quercetin has been investigated on proinflammatory cytokine expression and NO production, focusing on its effects on the MAP kinases and the NF-B signal transduction pathways in LPS-stimulated RAW 264.7 cells by using RT-PCR and immunoblotting. Quercetin strongly reduced activation of phosphorylated ERK kinase and p38 MAP kinase but not JNK MAP kinase by LPS treatment. In addition, quercetin treatment inhibited NF-B activation through stabilization of the NF-B/IB complex and IB degradation and proinflammatory cytokines and NO/iNOS expression. Quercetin may exert its anti-inflammatory and immunomodulatory properties in the effect molecules such as proinflammatory cytokines and NO/iNOS by suppressing the activation of ERK and p38 MAP kinase, and NF-B/IB signal transduction pathways.